US4835251A - Method of chain combination - Google Patents

Method of chain combination Download PDF

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US4835251A
US4835251A US06/877,819 US87781986A US4835251A US 4835251 A US4835251 A US 4835251A US 87781986 A US87781986 A US 87781986A US 4835251 A US4835251 A US 4835251A
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chain
relaxin
human relaxin
human
analog
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John P. Burnier
Paul D. Johnston
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Genentech Inc
BAS Medical Inc
Genetech Inc
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Genetech Inc
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Priority to US06/877,819 priority Critical patent/US4835251A/en
Priority to PH35432A priority patent/PH23407A/en
Priority to HU872804A priority patent/HU205948B/hu
Priority to IL82921A priority patent/IL82921A/xx
Priority to DK198703130A priority patent/DK173649B1/da
Priority to KR1019870006320A priority patent/KR960007604B1/ko
Priority to ZA874482A priority patent/ZA874482B/xx
Priority to DE8787305508T priority patent/DE3783273T2/de
Priority to PT85134A priority patent/PT85134B/pt
Priority to IE165587A priority patent/IE59683B1/en
Priority to EP87305508A priority patent/EP0251615B1/en
Priority to AU74620/87A priority patent/AU617291B2/en
Priority to JP62156391A priority patent/JP2529693B2/ja
Priority to CN87104447A priority patent/CN1029784C/zh
Priority to CA000540413A priority patent/CA1336223C/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/64—Relaxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/04—Drugs for genital or sexual disorders; Contraceptives for inducing labour or abortion; Uterotonics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S930/00—Peptide or protein sequence
    • Y10S930/01—Peptide or protein sequence
    • Y10S930/26—Containing cys-cys disulfide bridge between nonadjacent cysteine residues

Definitions

  • This invention provides a method for the combination of human relaxin A- and B-chains or human relaxin A- and B-chain analogs to produce useful yields of biologically active human relaxin or human relaxin analog.
  • the invention comprises combining the reduced human relaxin A- and B-chains or analogs thereof under conditions which include a pH greater than about 7.0 and which are mildly denaturing with respect to the human relaxin B-chain. These conditions provide a milieu for formation of biologically active human relaxin or an analog thereof by maintaining the mixture at a temperature of from about 15° C. to 30° C. with gradual exposure to air oxygen over the course of the reaction.
  • This invention also provides biologically active analogs of human relaxin.
  • This invention further provides a method of effecting parturition using human relaxin or an analog thereof as the sole active agent.
  • Human relaxin is an ovarian peptide responsible for remodelling the reproductive tract before parturition, thus facilitating the birth process. Hisaw, F. L., Pros. Soc. Exp. Bio. Med. 23, 661-663 (1926); Schwabe, C. et al. Biochem. Biophy. Res. Comm. 75, 503-570 (1977); James, R. et al., Nature 267, 544-546 (1977). While predominantly a hormone of pregnancy, relaxin has also been detected in the non-pregnant female as well as in the male. Bryant-Greenwood, G. D., Endocrine Reviews 3, 62-90 (1982) and Weiss, G., Ann. Rev. Physio. 46, 43-52 (1984).
  • the amino acid sequences of relaxim from pig, rat, tiger shark, dogfish shark and human have been established.
  • the hormone consists of two peptide chains, referred to as A and B, joined by disulfide bonds with an intro-chain disulfide loop in the A-chain in a manner analogous to that of insulin.
  • a and B two peptide chains
  • disulfide bonds with an intro-chain disulfide loop in the A-chain in a manner analogous to that of insulin.
  • a surprising and important difference between relaxin and most other peptide hormones, including insulin is the considerable structural variation between species.
  • pig, rat and human relaxins differ in over 50% of amino acid positions. These differences explain the poor immunological cross-reactivity between relaxins of different species and also a number of the observed differences in their specific biological activity.
  • H2 relaxin In the case of human relaxin two separate gene sequences have been identified. Id. Only one of these genes (H2) is expressed in the ovary during pregnancy, and it is unclear whether the other gene is expressed at another tissue site, or whether it represents a pseudo-gene.
  • the two human relaxin genes show considerable nucleotide and amino acid homology to each other, particularly in the B and C peptide. However, there are some notable regions of sequence divergence, particularly in the amino terminal region of both A- and B-chains. See FIG. 1. The fact that H2 relaxin is synthesized and expressed in the ovary suggests that this is the sequence which is involved in the physiology of pregnancy. In a recent paper Johnston, P. D.
  • FIG. 1 compares the known amino acid sequences of relaxins from different species.
  • the cysteine residues and flanking glycine residues only the isoleucine at position 7 in the B-chain, the arginine at positions 12 and 16, and the leucine at position 32 have been conserved.
  • the cysteine residues are clearly essential to maintaining the overall disulfide bond configuration.
  • Blundell, T. et al. In: Bigazzi, M., Greenwood, F. C., Gaspari, F. (eds.) Biology of Relaxin and its Role in the Human, (Excerpta Medica, Amsterdam, 1983) pp. 14-21.
  • Another feature of the relaxin structure is the variation in length seen at the amino and carboxyl terminal regions of the B-chain, and to a lesser extent at the amino terminus of the A-chain.
  • Chance et al. U.S. Pat. No. 4,421,685 issued Dec. 20, 1983 disclose a method for producing insulin or an analog thereof by combining the S-sulfonated form of the insulin A- and B-chains with a thiol reducing agent in an aqueous medium under controlled pH and temperature so as to carry out the reduction and oxidation reactions in a single step.
  • This method while presented as an improvement over the previously mentioned insulin synthesis, was found not to be applicable to the synthesis of human relaxin.
  • Another aspect of the invention is to produce biologically active analogs of human relaxin.
  • Yet another aspect of the invention is the use of human relaxin or an analog thereof to effect parturition.
  • the present invention is directed to a method of combining a human relaxin A-chain or analog thereof and a human relaxin B-chain or analog thereof to produce biologically active human relaxin or a human relaxin analog.
  • the method comprises combining the reduced human relaxin A-chain or analog and the reduced human relaxin B-chain or analog under conditions which include a pH greater than about 7.0, that is mildly denaturing with respect to the human relaxin B-chain and carrying out the reaction with gradual exposure to air oxygen over the course of the reaction.
  • FIG. 1 shows the lack of homology of human relaxin H1 and H2 with human insulin and procine (P) and rat (R) relaxins.
  • the numbering system is relative to H2 relaxin.
  • the disulfides for the relaxins are: A10-A15, A11-B11 and A24-B23.
  • FIG. 2(a) shows the HPLC time course for H2(B33 A24) B Lys 4 B Ala 25 and FIG. 2(b) for H2(B33 A24) profile of the in vitro chain combination reaction.
  • the time course of the combination reaction showing the formation of human relaxin analog or human relaxin and a potentially important intramolecular oxidized intermediate of the A-chain is described.
  • the conversion of gene-2 A- and B-chain peptides to H2 human relaxin analog and H2 human relaxin is delineated..
  • Chromatography is effected using a Synchropak RP-C4 (4.6 ⁇ 250 mm; 300 ⁇ ) with a linear gradient of acetonitrile (15 ⁇ 60% in 500 minutes) in a 0.05% TFA, H20 buffer running at 1 ml/min.
  • FIG. 4 shows the modified Bishop score in primate following administration of various doses of human relaxin analogs, H2(B2-33 A24) B Lys 4 B Ala 25 and H2(B33 A24) B Lys 4 B Ala 25 .
  • human relaxin or “human relaxin analog” refers to a functional protein that is known to remodel the reproductive tract to facilitate the birth process. Remodeling of the reproductive tract is understood to include such physiological actions as ripening of the cervix; thickening of the endometrium of the pregnant uterus as well as increased vascularization to this area; and, an effect on collagen synthesis.
  • Human relaxin has also been found in the female breast and may be associated with lactation. Human relaxin has also been found in human seminal fluid and may enhance the mobility of human spermatozoa. Given the effect of relaxin on the cervix human relaxin may augment the ability of sperm to penetrate the human cervix. Human relaxin may improve skin elasticity given its effect on connective tissue.
  • Human relaxin analog in addition to the functional definition described above structurally is meant to include a number of proteins, each of which has the basic structure of human relaxin including an A- and B-chain.
  • the human relaxin analog differs from the naturally occurring human relaxin by substitution, deletion, addition or modification of one or more amino acid residues in either the A- and/or B-chain of human relaxin with the caveat that biological relaxin-like activity is retained.
  • Examples of such human relaxin analogs include, but are not limited to: a full length A-chain and carboxy terminal shortened B-chain; H1(B2-27 A24) B Ala 25 ; H2(B2-25 A24); H2(B33 A24); H2(B33 A24) B Lys 4 B Ala 25 ; H2 (B2-33 A24) B Lys 4 B Ala 25 ; H2(B2-33 A24) A pyro-Glu 1 B Lys 4 B Ala 25 ; and H2(B33 A24) A pyro-Glu 1 B Lys.sup. 4 B Ala 25 .
  • H1, H2 refer to the two human genes which encode human relaxin.
  • a and B refer to the respective chains of human relaxin.
  • the numbers following A or B refer to the length of the chain, i.e. number of amino acids comrising the A- or B-chain.
  • Amino acids are designated by their customary three letter notation. The subscript preceding the amino acid designates the A- or B-chain in which the amino acid is located while the superscript following the amino acid refers to the position in the chain.
  • the relaxin A- and B-chains or analog chains may be obtained by classical protein synthetic methods, including either solution or solid phase techniques, or using recombinant DNA technology or by preparation from natural human relaxin or a combination of the above, e.g., chemical synthesis of one chain and recombinant DNA production of the other.
  • the individual peptide chains were synthesized via solid phase synthetic methodology, Barany, G. and Merrifield, R. B. (1980) in The Peptides 2, 1-284. Gross, E. and Meienhofer, J. Eds. Academic Press, New York. Protected N-t-butyloxycarbonyl amino acids were purchased from Peninsula Laboratories Inc.
  • the following side chain protection was used; Arg, tosyl; Asn, xanthyl; Asp, benzyl ester; Cys, methoxybenzyl; Gln, xanthyl; Glu, benzyl ester; His, tosyl; Lys, o-chlorobenzyloxycarbonyl; Ser, benzyl; Thr, benzyl; Tyr, 2,6-dichlorobenzyl.
  • the first amino acids were esterified onto chloromethylpolystyrene (1% divinylbenzene) with potassium fluoride in dimethylformamide. Substitution levels were 0.6 meq/gm.
  • amino acids were coupled with dicyclohexylcarbodiimide (distilled) in dichloromethane.
  • Arginine, asparagine, glutamine, leucine, and cysteine residues were coupled in 50/50 methylenechloride/dimethylformamide. Removal of the t-butyloxycarbonyl groups was accomplished with 45% trifluoroacetic acid, 5% anisole 5% ethanedithiol, and 45% methylenechloride. Neutralization prior to couplings was done with 10% triethylamine in methylenechloride.
  • the crude peptides were dissolved in 100 mmol dithiothreitol and then diluted into a large volume of 10% aqueous acetonitrile 0.1% trifluoroacetic acid. These solutions were loaded onto 5 ⁇ 55 cm columns packed with Vydac C18 (300 ⁇ 15-20 micron), washed with 0.1% aqueous trifluoroacetic acid, and eluted with a gradient of acetonitrile. The peptide fractions were pooled, lyophilized, and analyzed by amino acid composition, sequencing and analytical reverse phase HPLC. The cysteine residues were not protected by sulfonation or other derivatizations, Means, G. E. and Feeney, R.
  • the combination of human relaxin A- and B-chain or analog A- and B-chain to form human relaxin or a human relaxin analog can be achieved over a wide range of ratios of one chain relative to the other.
  • the combination is inherently limited by the chain, whether A or B, present in the lesser quantity.
  • Excess B-chain inhibits chain combination, while molar amounts of A-chain either in a slight excess or equal to B-chain are preferable.
  • the customary ratio of A-chain to B-chain, on a weight basis is from about 1:0.5 to about 3.0:1.
  • the protein concentration in the reaction medium is the protein concentration in the reaction medium.
  • the process can be successfully carried out over a wide range of protein concentrations. Generally, however, the protein concentration will range from about 0.1 to about 10 mg. per ml. of reaction medium. Preferably, the protein concentration will be in the range of from about 0.5 to about 5 mg.per ml. Again, it has been discovered, within this latter range, that the optimal protein concentration varies depending upon the human relaxin produced.
  • the process of this invention is carried out in an aqueous medium.
  • the pH of the medium measured at room temperature generally will range from about 7.0 to about 12. Preferably, it will be from about 7.5 to about 11.0 and optimally will be maintained within the range of from about 8.0 to about 10.6.
  • the pH of the medium may be maintained in the desired range by addition of a suitable suffering agent.
  • Typical buffering agents are, for example, glycinate, carbonate, tris (hydroxymethyl) aminomethane, pyrophosphate and other like agents which affect pH control within the aforedescribed range.
  • the common and preferred buffering agent is tris (hydroxymethyl) aminomethane (pH 8 to 9) and glycinate (pH 9.5 to 11.0).
  • the mixing reaction is carried out at a temperature of from about 15° C. to about 30° C. and preferably from about 20° C. to about 25° C. and most preferably at room temperature.
  • the concentration of buffering agent generally ranges from about 0.025M to about 0.2M. Preferably, the concentration is from about 0.05M to about 0.15M, and, most preferably about 0.1M.
  • One of the conditions of the inventive method is that it be carried out in an environment in which the exposure to air oxygen may be controlled. It was found that controlled oxidation could be achieved by N 2 purge of all solutions initially; that the reaction be N 2 purged in a closed container at the start of the reaction; and that while in a closed container the reaction be exposed to air directly by opening of the container or to oxygen by bubbling it into and through the medium.
  • Another condition of the inventive method is that the reaction be carried out under such conditions as to be mildly denaturing with respect to the human relaxin B-chain.
  • denaturing agents as urea, guanidine hydrochloride and other chaotropic agents, salts, detergents and organic solvent (acetonitrile, alcohols, dimethylformamide, etc.) known to the ordinarily skilled artisan could be used.
  • organic solvent acetonitrile, alcohols, dimethylformamide, etc.
  • urea and acetonitrile and a few percent based on volume (less than 10%) of organic solvents render the conditions mildly denaturing to the human relaxin B-chain.
  • the human relaxin A- and B-chains are brought together in the appropriate aqueous medium in free-cysteine reduced forms.
  • the reactions were started by first adding A-chain followed by B-chain from fresh 5 mg/ml stocks in H 2 O at pH2. The pH is adjusted to the appropriate value with NaOH.
  • Each reaction is monitored by RP-4 reverse phase analytical HPLC Snyder, L. R. and Kirkland, J. J. in Introduction to Modern Liquid Chromatography (1979), for maximal formation of recombined relaxin and stopped by addition of glacial acetic acid to pH 4.
  • the mixture is then centrifuged at 16,318 ⁇ g for 30 minutes and the supernatant purified by preparative reverse phase HPLC. Id.
  • the human relaxin or relaxin analog product can be isolated by any of a wide variety of methods, all of which are recognized in the field of protein isolation.
  • the most commonly employed techniques for relaxin purification are chromatographic techniques. These are readily applicable in recovering human relaxin from the process of this invention. These can include gel filtration, ion-exchange chromatography or reverse phase HPLC.
  • An example of a purification scheme was to load the reaction supernatants totaling 0.5 to 1 gram of relaxin peptide on a (15-20 micron) Vydac C4 300 ⁇ (5 ⁇ 50 cm) column. Purification was achieved using a 20 to 40% acetonitrile gradient (0.5% change per minute) in H 2 O, 0.1% TFA. The flow rate was 20 ml/min and 1.0 minute fractions were collected. These were monitored isocratically 25% acetonitrile, H 2 O, 0.1% TFA on an analytical (5 micron) Vydac C4 300 ⁇ (4.6 ⁇ 250 mm) column. Fractions containing major products were pooled and lyophilized for further analysis.
  • the human relaxin and human relaxin analogs were tested in bioassays.
  • the murine pubic symphysis assay uses Charles River albino CFW female mice weighing 18-20 g., J. St. Louis, Can. J. Physiol. Pharmacol. 59, 507-512 (1981).
  • Estradiol priming solution is 5 ⁇ g estradiol 17 ⁇ -cyclopentylpropionate in 0.1 ml peanut oil.
  • Human relaxin dose solutions are at concentrations of 2.5, 5.0, 10.0, 20.0, 40.0, and 80.0 ⁇ g/ml in a 1% water solution of benzopurpurine 4B.
  • mice When mice have been housed for at least six days in quarantine and weigh 18-20 g, each one is given a subcutaneous injection of estradiol priming solution. Exactly seven days later mice are injected subcutaneously with 0.2 ml of the appropriate human relaxin dose solution. Between 18 and 20 hours after the human relaxin injection the mice were sacrificed by cervical dislocation. The interpubic ligament is exposed and measured with a micrometer.
  • Estradiol priming solution is 200 ⁇ g estradoil 17 ⁇ -cyclopentylpropionate in 0.1 ml peanut oil.
  • Human relaxin stock solution is at a concentration of 0.1 mg/ml in sterile injection water (Invenex).
  • Invenex sterile injection water
  • Each tissue is suspended in a jacketed water bath kept at 37° C. containing 35 ml of aerated Holmans Ringer Solution.
  • the tissue is caused to contract by replacing the Ringer solution with one in which 20 percent of the sodium chloride is replaced with an equimolar amount of potassium chloride.
  • a concentration of relaxin is added to the bath.
  • doses of porcine relaxin added to the bath are 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, and 12.8 ⁇ g relaxin per 35 ml in the bath.
  • Human relaxin's induction of cervical ripening in a nonhuman primate pregnancy was tested. Human relaxin was tested in time-mated Rhesus monkeys at gestational ages from 130 to 160 days. Term gestation in the colony is 168 ⁇ 6 days.
  • the animals were adapted to wearing jackets with tethers. The tethered jackets permitted continuous access to indwelling catheters placed either in the femoral vessels or in the carotid and internal jugular vessels. In most animals a pressure transducer was placed within the amniotic sac and electrodes connected to the uterine surface allowing measurement of uterine contractile activity.
  • Relaxin was administered as a continuous IV infusion over one hour, with blood samples obtained before, during and after the infusion at intervals from 15 minutes through the first hour following the infusion and then regularly for 24 hours. Doses of relaxin ranged from 10 ⁇ g to 100 ⁇ g, and the cervix was scored for texture, effacement, position, dilation, and fetal position relative to the cervix, as well as the quality of the uterine lower segment. In most cases, two observers examined each cervix and scored them independently. Controls were monkeys operated upon in similar manner and infused with saline (one) or monkeys maintained in the colony but not operated upon with cervical exams performed at weekly intervals.
  • the gels are visualized by Coomassie Blue (gel soaked 1.5 hrs in 10ml acetic acid plus 90ml (0.25% weight/volume) Coomassie Blue R-250 in 25% ethanol), silver stain (Oakley, B. R. et al. Analytical Biochem. (1980) 105, 361-363), after fixing and destaining in a solution of 0.2% formaldehyde, 20% ethanol, and 6.2% acetic acid (all volume/volume) for 15-30 minutes and Western analysis (Towbin, H. et al. PNAS (1979) 76; 4350-4354).
  • Antibodies specific for the A- and B-chains were raised by immunizing New Zealand white rabbits with the free peptides either alum precipitated or in Freund's adjuvant as described in Eddie, L. W. et al. (1986) The lancet 1, 1344-1346.
  • the titers were essentially the same and antisera from each immunization was pooled with bb titer for the A-chain and ccc titer for the B-chain. These were used at 500 fold dilution in the incubation against the nitrocellulose filters 2 hours to overnight. The washed filters are then incubated against 125I-protein A for 2 hours, dried and placed against X-ray film.
  • Reduced relaxin A-chain (10 mg) was added as a reduced solid lyophilized powder to the reaction mixture.
  • Reduced relaxin B-chain (5.63 mg) was also added as a solid lyophilized powder.
  • the A and B relaxin chain solutions were combined in a 10 ml vial at room temperature ( ⁇ 25° C.) by first adding the relaxin A-chain followed by relaxin B-chain as solid lyophilized powders from above. The pH was adjusted to 10.5 using NaOH. The reaction was monitored by RP-4 reverse phase analytical HPLC for maximal formation of recombined relaxin. Due to the insolubility of the natural form of H2 B-chain, its recombination with A-chain required the following conditions in accord with the invention; final reaction 0.1M glycine, pH 10.5, 1mM EDTA, 2.5mM DTT, 3% 1-propanol, 3% acetonitrile, and 1M urea.
  • the mixture was purified by preparative RP-4 reserve phase HPLC (1 ⁇ 25cm) RP-4 Synchropak 300 A. The peak was pooled and used directly out of HPLC solvent. The human relaxin was judged to be quite pure by polyacrylamide gel electrophoresis, amino acid analysis HPLC, amino terminal sequencing and bioassay.
  • Reduced human relaxin A-chain (200 mg) was dissolved in 40 ml of H 2 O (pH 2.0).
  • Reduced human relaxin B-chain (B33 B Lys 4 Ala 25 ) (100 mg) was dissolved in 20 ml of H 2 O (pH 2).
  • the A- and B-chain analog solutions were combined in a 165 ml vial at room temperature ( ⁇ 25° C.) by first adding the A-chain followed by modified B-chain from the foregoing fresh stocks in H 2 O, pH2. The pH was adjusted to about 8.0 with NaOH. The reaction was monitored by RP-4 reverse phase HPLC for maximal formation of combined human relaxin analog.
  • human relaxin analog the following conditions in accord with the instant invention were used; reaction B 0.1M tris, pH 8.0, 1 mM EDTA, 2 mM DTT, 24° C. The reaction was stirred vigorously open to the air. The combination reaction was stopped by addition of glacial acetic acid to pH 4.
  • the mixture was purified by preparative HPLC Vydac C4 300 ⁇ (5 ⁇ 50 cm).
  • the human relaxin analog peak (elution volume, about 140 ml) was pooled and lyophilized with a recovery of 35 mg of relaxin, or 20.3% incorporation of B-chain.
  • the human relaxin analog was judged to be quite pure by polyacrylamide gel electrophoresis, amino acid analysis, amino terminal sequencing, HPLC (see FIG. 2a.) and the bioassay.
  • Reduced human relaxin A-chain (41.5 mg) was dissolved in 8 ml of H 2 O (pH 2.0).
  • the A- and B-chain analog solutions were combined in a 33 ml vial at room temperature ( ⁇ 25° C.) by first adding the A-chain followed by modified B-chain from the foregoing fresh stocks in H 2 O, pH2. The pH was adjusted to 8.0 with NaOH. The reaction was monitored by RP-4 reverse phase HPLC for maximal formation of combined human relaxin analog.
  • this relaxin analogue the following conditions in accord with the instant invention were used: reaction D 0.1M Tris, pH 8, 25° C.; purged with N 2 and stirred under N 2 atmosphere for the first 1-2 hours. The reaction was then stirred vigorously open to the air. The combination reaction was stopped by addition of glacial acetic acid to pH4.
  • the mixture was purified by preparative HPLC Vydac C4 300 ⁇ (5 ⁇ 80 cm).
  • the relaxin analog peak (elution volume, about 140 ml) was pooled and lyophilized with a recovery of 16 mg of human relaxin analog, or 40.3% incorporation of B-chain.
  • the relaxin analog was judged to be quite pure by polyacrylamide gel electrophoresis, amino acid analysis, amino terminal sequencing, HPLC (see FIG. 2a.) and the bioassay.
  • Reduced human relaxin A-chain (41.5) was dissolved in 8 ml of H 2 O (pH 2.0).
  • the A- and B-chain analog solutions were combined in a 33 ml vial at room temperature ( ⁇ 25° C.) by first adding the A-chain followed by modified B-chain from the foregoing fresh stocks in H 2 O, pH2. The pH was adjusted to 8.0 with NaOH. The reaction was monitored by RP-4 reverse phase HPLC for maximal formation of combined human relaxin analog.
  • this relaxin analogue the following conditions in accord with the instant invention were used: reaction D 0.1M Tris, pH 8, 25° C.; purged with N 2 and stirred under N 2 atmosphere for the first 1-2 hours. The reaction was then stirred vigorously open to the air. The combination reaction was stopped by addition of glacial acetic acid to pH4.
  • the mixture was purified by preparative HPLC Vydac C4 300 ⁇ (5 ⁇ 80 cm).
  • the relaxin analog peak (elution volume, about 140 ml) was pooled and lyophilized with a recovery of 7.5 mg of human relaxin analog, or 18.9% incorporation of B-chain.
  • the relaxin analog was judged to be quite pure by polyacrylamide gel electrophoresis, amino acid analysis, amino terminal sequencing, HPLC (see FIG. 2a.) and the bioassay.
  • Reduced human relaxin A-chain (200 mg) was dissolved in 40 ml of H 2 O (pH 2.0).
  • Reduced human relaxin B-chain (B33 B Lys 4 B Ala 25 ) (100 mg) was dissolved in 20 ml of H 2 O (pH 2).
  • the A- and B-chain analog solutions were combined in a 165 ml vial at room temperature ( ⁇ 25° C.) by first adding the A-chain followed by modified B-chain from the foregoing fresh stocks in H 2 O, pH2. The pH was adjusted to about 8.0 with NaOH. The reaction was monitored by RP-4 reverse phase HPLC for maximal formation of combined human relaxin analog.
  • reaction B 0.1M sodium glycinate, pH 8.0, 1 mM EDTA, 2 mM DTT, 24° C. The reaction was stirred vigorously open to the air. The combination reaction was stopped by addition of glacial acetic acid to pH4.
  • the mixture was purified by preparative HPLC Vydac C4 300 ⁇ (5 ⁇ 50 cm).
  • the human relaxin analog peak (elution volume, about 140 ml) was pooled and lyophilized with a recovery of 16 mg of relaxin, or 9.3% incorporation of B-chain.
  • the human relaxin analog was judged to be quite pure by polyacrylamide gel electrophoresis, amino acid analysis, amino terminal sequencing, HPLC (see FIG. 2a.) and the bioassay.
  • the rat uterine contractility in vitro bioassay measures human relaxin ability to relax smooth muscle in the presence of electrically stimulated contractions, J. St. Louis, (1981) Can. J. Physiol. Pharmacol. 59, 507-512.
  • the murine pubic symphysis ligament in vivo bioassay measures relaxin's remodeling effect on connective tissue, Steinetz, B. G. et al. (1960) Endocrinology 67, 102.
  • H2(B2-33 A24), H2(B32 A24) B Lys 4 B Ala 25 , H2(B33 A24) B Lys 4 B Ala 25 , and the pyro-Glu forms of the foregoing H2(B2-33 A24) B Lys 4 B Ala 25 A pyro-Glu 1 and H2(B33 A24) B Lys 4 B Ala 25 A pyro-Glu 1 indicated biological activity in both the MPS and RUC assays.
  • the MPS data for a human relaxin analog and human relaxin is shown in FIG. 3.
  • the MPS does responses indicate equipotence for natural human relaxin and H2(B2-33 A24) B Lys 4 B Ala 25 , FIG. 3.
  • porcine relaxin appears to have a non-parallel response relative to the other relaxins. All of the human analogues and natural sequence human relaxin are essentially indistinguishable in the MPS bioassay.
  • the mean cervical change for 7 separate infusions of various doses was 3.7 units. These data are presented in FIG. 5, where the individual infusions at 100, 50 and 10 ⁇ g doses are shown, as well as an average of all other 10 ⁇ g infusions and the two kinds of controls. Note the non-infused control monkeys range from 2 to 15 determinations per point (except for two points each is representative of at least 4 monkeys, and the mean is plotted. In general, the cervix was more sensitive to lower doses of relaxin as gestation progressed, or to repeated administration of relaxin early in this time period.
  • the human relaxin and human relaxin analogs of the present invention can be formulated using known methods to prepare pharmaceutically useful compositions such that the human relaxin or analog thereof is combined with a pharmaceutically acceptable carrier.
  • Suitable vehicles and their formulation, including other necessary human proteins, e.g., human serum albumin, are described in standard formulation treatises e.g. Remington's Pharmaceutical Sciences by E. W. Martin.

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US06/877,819 US4835251A (en) 1986-06-23 1986-06-23 Method of chain combination
PH35432A PH23407A (en) 1986-06-23 1987-06-19 A method for the combination of human relaxin a and b-chain analogs
HU872804A HU205948B (en) 1986-06-23 1987-06-19 Process for producing human relaxin and theyr analogues of the same activity
IL82921A IL82921A (en) 1986-06-23 1987-06-19 Method of chain combination of human relaxin
DK198703130A DK173649B1 (da) 1986-06-23 1987-06-19 Fremgangsmåde til kædekombination til fremstilling af biologisk aktivt humant relaxin eller analoge deraf
EP87305508A EP0251615B1 (en) 1986-06-23 1987-06-22 Method of chain combination
ZA874482A ZA874482B (en) 1986-06-23 1987-06-22 Method of chain combination
DE8787305508T DE3783273T2 (de) 1986-06-23 1987-06-22 Methode fuer das zusammensetzen einer kette.
PT85134A PT85134B (pt) 1986-06-23 1987-06-22 Metodo para combinacao de cadeias de relaxina humana
IE165587A IE59683B1 (en) 1986-06-23 1987-06-22 Method of chain combination
KR1019870006320A KR960007604B1 (ko) 1986-06-23 1987-06-22 인체 렐락신 또는 그 유사체를 제조하는 방법
CA000540413A CA1336223C (en) 1986-06-23 1987-06-23 Method of chain combination
JP62156391A JP2529693B2 (ja) 1986-06-23 1987-06-23 鎖の結合法
CN87104447A CN1029784C (zh) 1986-06-23 1987-06-23 链结合的方法
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US5166191A (en) * 1991-08-19 1992-11-24 Genentech, Inc. Use of relaxin in cardiovascular therapy
US5191063A (en) * 1989-05-02 1993-03-02 University Of Medicine And Dentistry Of New Jersey Production of biologically active polypeptides by treatment with an exogenous peptide sequence
US5464756A (en) * 1989-05-04 1995-11-07 Genentech Processes and compositions for the isolation of human relaxin
US5478807A (en) * 1991-08-19 1995-12-26 Genentech, Inc. Use of relaxin in the treatment of bradycardia
US5994148A (en) * 1997-06-23 1999-11-30 The Regents Of University Of California Method of predicting and enhancing success of IVF/ET pregnancy
US6048544A (en) * 1995-11-17 2000-04-11 Yue; Samuel K. Method of collagen therapy using relaxin
US6251863B1 (en) 1998-09-08 2001-06-26 Samuel K. Yue Method of preventing and treating symptoms of aging and neurodegenerative dysfunctions with relaxin
US20040086509A1 (en) * 1996-08-02 2004-05-06 Zymogenetics, Inc. Testis-specific insulin homolog polypeptides
US20050032683A1 (en) * 2000-10-04 2005-02-10 Amento Edward P. Methods of modulating apoptosis by administration of relaxin agonists or antagonists
US20050186526A1 (en) * 2002-11-01 2005-08-25 Bas Medical, Inc. Methods and systems for enabling and stabilizing tooth movement
US20060052304A1 (en) * 2004-09-02 2006-03-09 Bas Medical, Inc. Method for remodeling bone and related sutures
US20060247172A1 (en) * 2004-04-30 2006-11-02 Bas Medical, Inc. Methods and compositions for control of fetal growth via modulation of relaxin
US20100286046A1 (en) * 2006-04-11 2010-11-11 Jeffrey A Medin Modified h2 relaxin for tumor suppression
WO2010140060A2 (en) 2009-06-01 2010-12-09 Chemical & Biopharmaceutical Laboratories Of Patras S.A. Peptide synthesis
WO2012024452A2 (en) 2010-08-17 2012-02-23 Ambrx, Inc. Modified relaxin polypeptides and their uses
WO2013177529A1 (en) * 2012-05-24 2013-11-28 Angion Biomedica Corp. Relaxin-like compounds and uses thereof
EP2946788A1 (en) 2014-05-23 2015-11-25 Immundiagnostik AG Method and composition for treating heart failure with preserved ejection fraction
US9381231B2 (en) 2012-10-09 2016-07-05 University Of Florida Research Foundation, Inc. Use of relaxin to restore maternal physiology in pregnancies conceived by assisted reproductive technologies
WO2017079746A2 (en) 2015-11-07 2017-05-11 Multivir Inc. Methods and compositions comprising tumor suppressor gene therapy and immune checkpoint blockade for the treatment of cancer
WO2018111902A1 (en) 2016-12-12 2018-06-21 Multivir Inc. Methods and compositions comprising viral gene therapy and an immune checkpoint inhibitor for treatment and prevention of cancer and infectious diseases
WO2020036635A2 (en) 2018-03-19 2020-02-20 Multivir Inc. Methods and compositions comprising tumor suppressor gene therapy and cd122/cd132 agonists for the treatment of cancer
WO2021022139A1 (en) 2019-07-31 2021-02-04 Eli Lilly And Company Relaxin analogs and methods of using the same
WO2021113644A1 (en) 2019-12-05 2021-06-10 Multivir Inc. Combinations comprising a cd8+ t cell enhancer, an immune checkpoint inhibitor and radiotherapy for targeted and abscopal effects for the treatment of cancer

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US5911997A (en) * 1995-06-07 1999-06-15 Connetics Corporation Relaxin-like factor and methods and uses thereof
AU2003903124A0 (en) * 2003-06-20 2003-07-10 Mark Del Borgo Analogues of heteromeric proteins
GR1006759B (el) * 2008-12-22 2010-04-20 Χημικα Και Βιοφαρμακευτικα Εργαστηρια Πατρων Αε (Cbl-Patras) Εξελιγμενη χημικη μεθοδος συνθεσης της ανθρωπινης ρηλαξινης
GR1007010B (el) * 2009-10-08 2010-10-07 Χημικα Και Βιοφαρμακευτικα Εργαστηρια Πατρων Αε (Cbl-Patras), Ινσουλινοειδη πεπτιδια
JP6289937B2 (ja) * 2014-02-27 2018-03-07 学校法人東海大学 リラキシンの製造方法
WO2021226439A2 (en) * 2020-05-08 2021-11-11 President And Fellows Of Harvard College Engineered relaxins and methods of use thereof

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Cited By (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5191063A (en) * 1989-05-02 1993-03-02 University Of Medicine And Dentistry Of New Jersey Production of biologically active polypeptides by treatment with an exogenous peptide sequence
US5464756A (en) * 1989-05-04 1995-11-07 Genentech Processes and compositions for the isolation of human relaxin
US5478807A (en) * 1991-08-19 1995-12-26 Genentech, Inc. Use of relaxin in the treatment of bradycardia
US5166191A (en) * 1991-08-19 1992-11-24 Genentech, Inc. Use of relaxin in cardiovascular therapy
US6048544A (en) * 1995-11-17 2000-04-11 Yue; Samuel K. Method of collagen therapy using relaxin
US7070774B2 (en) * 1996-08-02 2006-07-04 Zymogenetics, Inc. Antibodies that bind testis-specific insulin homolog polypeptides
US20040086509A1 (en) * 1996-08-02 2004-05-06 Zymogenetics, Inc. Testis-specific insulin homolog polypeptides
US20060150267A1 (en) * 1996-08-02 2006-07-06 Zymogenetics, Inc. Methods of using testis-specific insulin homolog polypeptides
US6660531B2 (en) 1997-06-23 2003-12-09 The Regents Of The University Of California Relaxin levels corrlelated to IVF/ET pregnancy success
US5994148A (en) * 1997-06-23 1999-11-30 The Regents Of University Of California Method of predicting and enhancing success of IVF/ET pregnancy
US6251863B1 (en) 1998-09-08 2001-06-26 Samuel K. Yue Method of preventing and treating symptoms of aging and neurodegenerative dysfunctions with relaxin
US20050032683A1 (en) * 2000-10-04 2005-02-10 Amento Edward P. Methods of modulating apoptosis by administration of relaxin agonists or antagonists
US9534034B2 (en) * 2000-10-04 2017-01-03 Molecular Medicine Research Institute Methods of modulating apoptosis by administration of relaxin agonists or antagonists
US7833526B2 (en) 2000-10-04 2010-11-16 Molecular Medicine Research Institute Methods of modulating apoptosis by administration of relaxin agonists or antagonists
US20120238499A1 (en) * 2000-10-04 2012-09-20 Molecular Medicine Research Institute Methods of modulating apoptosis by administration of relaxin agonists or antagonists
US8119136B2 (en) 2000-10-04 2012-02-21 Molecular Medicine Research Institute Methods of modulating apoptosis by administration of relaxin agonists or antagonists
US20080085275A1 (en) * 2000-10-04 2008-04-10 Molecular Medicine Research Institute Methods of modulating apoptosis by administration of relaxin agonists or antagonists
US20110059108A1 (en) * 2000-10-04 2011-03-10 Molecular Medicine Research Institute Methods of modulating apoptosis by administration of relaxin agonists or antagonists
US20050186526A1 (en) * 2002-11-01 2005-08-25 Bas Medical, Inc. Methods and systems for enabling and stabilizing tooth movement
US8026215B2 (en) 2004-04-30 2011-09-27 Corthera, Inc. Methods and compositions for control of fetal growth via modulation of relaxin
US7553813B2 (en) 2004-04-30 2009-06-30 Corthera, Inc. Methods and compositions for control of fetal growth via modulation of relaxin
US20060247172A1 (en) * 2004-04-30 2006-11-02 Bas Medical, Inc. Methods and compositions for control of fetal growth via modulation of relaxin
US20060247163A1 (en) * 2004-04-30 2006-11-02 Bas Medical, Inc. Methods and compositions for control of fetal growth via modulation of relaxin
US20080108572A1 (en) * 2004-04-30 2008-05-08 Bas Medical, Inc. Methods and compositions for control of fetal growth via modulation of relaxin
US20060052304A1 (en) * 2004-09-02 2006-03-09 Bas Medical, Inc. Method for remodeling bone and related sutures
US8445635B2 (en) 2006-04-11 2013-05-21 Armour Therapeutics Inc. Modified H2 relaxin for tumor suppression
US20100286046A1 (en) * 2006-04-11 2010-11-11 Jeffrey A Medin Modified h2 relaxin for tumor suppression
US20110039778A1 (en) * 2009-06-01 2011-02-17 Chemical & Biopharmaceutical Laboratories Of Patras S.A. Peptide Synthesis
WO2010140060A2 (en) 2009-06-01 2010-12-09 Chemical & Biopharmaceutical Laboratories Of Patras S.A. Peptide synthesis
US8785384B2 (en) * 2009-06-01 2014-07-22 Chemical & Biopharmaceutical Laboratories of Patras Peptide synthesis
WO2012024452A2 (en) 2010-08-17 2012-02-23 Ambrx, Inc. Modified relaxin polypeptides and their uses
EP4302783A2 (en) 2010-08-17 2024-01-10 Ambrx, Inc. Modified relaxin polypeptides and their uses
WO2013177529A1 (en) * 2012-05-24 2013-11-28 Angion Biomedica Corp. Relaxin-like compounds and uses thereof
US9381231B2 (en) 2012-10-09 2016-07-05 University Of Florida Research Foundation, Inc. Use of relaxin to restore maternal physiology in pregnancies conceived by assisted reproductive technologies
US10434146B2 (en) 2012-10-09 2019-10-08 University Of Florida Research Foundation, Inc. Use of relaxin to restore maternal physiology in pregnancies conceived by assisted reproductive technologies
WO2015177378A1 (en) 2014-05-23 2015-11-26 Immundiagnostik Ag Method and composition for treating heart failure with preserved ejection fraction
EP2946788A1 (en) 2014-05-23 2015-11-25 Immundiagnostik AG Method and composition for treating heart failure with preserved ejection fraction
WO2017079746A2 (en) 2015-11-07 2017-05-11 Multivir Inc. Methods and compositions comprising tumor suppressor gene therapy and immune checkpoint blockade for the treatment of cancer
WO2018111902A1 (en) 2016-12-12 2018-06-21 Multivir Inc. Methods and compositions comprising viral gene therapy and an immune checkpoint inhibitor for treatment and prevention of cancer and infectious diseases
WO2020036635A2 (en) 2018-03-19 2020-02-20 Multivir Inc. Methods and compositions comprising tumor suppressor gene therapy and cd122/cd132 agonists for the treatment of cancer
WO2021022139A1 (en) 2019-07-31 2021-02-04 Eli Lilly And Company Relaxin analogs and methods of using the same
WO2021113644A1 (en) 2019-12-05 2021-06-10 Multivir Inc. Combinations comprising a cd8+ t cell enhancer, an immune checkpoint inhibitor and radiotherapy for targeted and abscopal effects for the treatment of cancer

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EP0251615A2 (en) 1988-01-07
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DK173649B1 (da) 2001-05-21
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HU205948B (en) 1992-07-28
PT85134B (pt) 1990-03-30
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DE3783273D1 (de) 1993-02-11
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JPS6366198A (ja) 1988-03-24
JP2529693B2 (ja) 1996-08-28
KR960007604B1 (ko) 1996-06-07
KR880000463A (ko) 1988-03-26
CN87104447A (zh) 1988-02-10

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